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Abstract

In order to prepare protein extracts of Plasmodium falciparum blood stages for western blot analysis, infected red blood cells (iRBC) need to be separated from uninfected red blood cells (uRBC) which make up the bulk of the parasite culture. Depending on the localisation of the parasite protein of interest, different methods are available to achieve this. If the protein is present within the parasite or is attached to a cellular structure of the iRBC cell, saponin can be used. This reagent lyses the membranes of infected and uninfected erythrocytes, the Maurer´s clefts (vesicular structures in the iRBC) and the parasitophorous vacuole membrane containing the parasite but leaves the parasite plasma membrane intact, providing a convenient procedure to isolate intact parasites without uRBCs. However, this method has the disadvantage that the host cell cytosol and the parasitophorous vacuole (PV) content of iRBCs are lost. If this has to be avoided, it is possible to use a Percoll gradient to separate intact iRBCs from uRBCs. Sequential treatment with Tetanolysin and saponin can then be used to selectively release the iRBC cytosol and the PV content from the parasite. These selective lysis methods are also suitable to determine the subcellular localisation of a protein of interest.

Centrifuge the lysate at 16,000 x g for 5 min to pellet the parasites
and repeatedly wash the pellet with 1x PBS until the supernatant shows
no red colour anymore.Note: Usually 3 washing steps are necessary.

Discard the supernatant and add 2-8 µl 25x protease inhibitor
cocktail. Depending on the size of the parasite pellet, resuspend the
pellet in 50-200 µl parasite lysis buffer. Note: In general 100 µl
parasite lysis buffer will work for 10 ml of a parasite culture >5%
parasitemia but this depends on the proportion of schizont stage
parasites as these stages contain a lot of DNA. Addition of DNase can be
used to reduce the viscosity of the extract.

The extracts can be stored at -20 °C if not used immediately for SDS-PAGE.

Centrifuge 10 ml of a Plasmodium falciparum culture (5-10%
parasitemia) at 500 x g for 5 min, discard the supernatant and resuspend
the pellet in 10 ml 1x PBS. Centrifuge again and remove the
supernatant.

Prepare the percoll gradient by adding first 500
µl 80% percoll solution to a 2 ml tube, then carefully layering 500 µl
60% percoll solution on top of the 80% percoll and finally layering
500 µl 40% percoll solution on top of the 60% percoll (Figure 1,
Percoll gradient). Note: Use the gradient immediately.

Resuspend the pellet with 200 µl 1x PBS and slowly pipette the parasite
solution on top of the gradient. Centrifuge immediately at 16,000 x g for 5 min. Note: Touch the side of the tube with the pipette tip while adding the parasites to avoid mixing of the solutions.

Carefully collect the desired parasite stage band into a new 1.5 ml
tube (see Figure 1 for selection of correct phase from the gradient) and
add 1 ml 1x PBS. Note: If too much percoll solution is transferred,
for instance if no clear iRBC phase was apparent (Figure 1A, scenario
3), the parasites may not pellet properly after the first washing step.
In this case only remove the top region after centrifugation of the
first wash, and replace again with 1x PBS to bring down the overall
percoll concentration of the solution. The iRBCs should then pellet
after in the next centrifugation step.

Centrifuge at 16,000 x g for 5 min and wash the parasite pellet at least 3x with 1 ml 1x PBS.Note: Use a fresh tube for each washing step.

Centrifuge the lysate at 16,000 x g for 5 min. Transfer the saponin
supernatant into a fresh tube (final extract of host cell cytosol and PV
content) and add 4 µl 25x protease inhibitor cocktail. The extract can
be stored at -20 °C if not used immediately for SDS-PAGE.

Wash the pellet with 1x PBS until the supernatant is clear.Note: In general 3 washing steps are necessary.

Add 4 µl 25x protease inhibitor cocktail to the pellet. Resuspend the
pellet in 100 µl parasite lysis buffer. The extract can be stored at -20
°C if not used immediately for SDS-PAGE.

Figure 1. Percoll gradient
and outcomes. A. Preparation/Set-up of a Percoll gradient without and
with parasite culture (top left). Three possible results after
centrifugation are shown under 'outcomes' (top right): 1) one band
consisting of mostly debris, merozoites and segmented schizonts at the
border between the 40% Percoll and the aqueous region (present above the
40% percoll after centrifugation) and one band above uRBC/ring stage
region consisting of younger schizonts and trophozoites (This is the
fraction to harvest.); 2) similar appearance to 1) but with multiple
bands below the debris/merozoites/segmented schizonts phase; these bands
usually consist of different stages of iRBCs and can all be collected;
3) no clearly defined band; in this case collect the indicated fraction
(from the mid 40% to the region just above the uRBCs and ring stage
phase, making sure not to disturb this phase). Although in this case the
stages may be too distributed to be seen as a clear band in the
gradient, this usually will still result in a good recovery once
pelleted and washed. B. Example of a purification of iRBCs infected with
PF13_0191-GFP parasites. GFP, PF13_0191-GFP fluorescence; DIC,
differential interference contrast; merge, both images merged.

Percoll gradient and sequential treatment with tetanolysin and saponin
to obtain extracts of the host cell cytosol and the PV content,
respectively.

Perform steps A2a-e.

Resuspend the
parasite pellet in 99 µl 1x PBS and add 1 µl tetanolysin (1 µg/ml).
Mix the solution immediately by flipping the tube with your finger.
Incubate at 37 °C for 30 min.Note: The tetanolysin activity may vary
with batch and depending on the number of freeze thaw cycles of the
aliquot used. If standardised results are needed, activity testing with
uRBCs can be conducted before each use. For this incubate serial
dilutions of tetanolysin with 20 µl of 70% haematocrit RBCs for 1 min at
37 °C followed immediately by centrifugation at 16,000 x g for 3 min to
assess lysis (pellet of uRBCs left). For the percoll pellet use 10x the
amount of tetanolysin required to just lyse all of the 20 µl uRBCs.
Note that the actual number of parasites obtained after percoll
purification also influences lysis efficiency.

Centrifuge the
lysate at 16,000 x g for 5 min. Transfer the tetanolysin supernatant
into a fresh tube (final extract of host cell cytosol) and add 4 µl 25x
protease inhibitor cocktail. The extract can be stored at -20 °C if not
used immediately for SDS-PAGE.

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